The lymphatic system is essential in mediating tissue fluid homeostasis, intestinal lipid uptake and immune surveillance. Lymphatic diseases including lymphedema occur from dysfunctional lymphatic networks and are a significant public health issue that may cause loss of function in the limbs. How dysfunctional lymphatics aggravate lymphatic diseases, and conversely, how dysfunctional lymphangiogenesis improves lymphatic disorders are poorly understood but important medically-relevant questions. Our long-term goal is to understand the cellular and molecular processes that control lymphangiogenesis and lymphatic function in order to better treat lymphatic diseases. The Neuropilin 2 (NRP2) receptor is unique in that it transmits both pro-lymphangiogenic signals from VEGF-C/D ligands in concert with VEGFR3 as well as suppressive signals from Semaphorin-3F (SEMA3F) via plexin receptors. Primary lymphedema patients have been found to have mutations in Foxc2, VEGFR3, or NRP2. Our recent publication showing prolonged lymphedema, persistent swelling, and insufficient lymphatic drainage in NRP2-deficient mice during acute inflammatory reactions provides a strong premise for this proposal. Our new mice which inducibly lack NRP2 in lymphatic endothelium have attenuated lymphangiogenesis. Additionally, new data demonstrates that Foxc2 negatively regulates NRP2 (as well as VEGFR3) expression. Together, these data suggest that NRP2 is necessary for normal resolution of extravasated fluid. We hypothesize that the NRP2 signaling axis can be manipulated for therapeutic gain in models of lymphatic dysfunction and chronic inflammation. We will test our hypothesis by pursuing three interrelated yet independent aims. Aim 1 will explore the mechanisms underlying NRP2 and VEGFR3 signaling in adult lymphangiogenesis in vivo using tissue-specific genetic ablation strategies and quantitative near infrared imaging to evaluate lymphatic drainage in the context of inflammation. Given that Foxc2 downregulates NRP2 but NRP2 is normally upregulated during pathological conditions, we will assess NRP2-mediated augmentation of lymphatic function in adult wild type and lymphatic-specific Foxc2 loss-of-function mice. Aim 2 will examine the molecular mechanisms regulating NRP2 expression in lymphatic endothelium. Correspondingly, lymphatic-specific Foxc2 gain-of-function mice exhibit reduced lymphatic function similar to lymphatic- specific NRP2-deficient mice. How NRP2 expression is regulated via Foxc2 is poorly understood but new data suggests that it may interfere with COUP-TFII activity. Aim 3 will investigate the therapeutic potential of inhibiting endogenous SEMA3F to treat lymphatic dysfunctions. Overall, this project is innovative as it will generate novel transgenic animal models, reveal distinct regulatory molecular mechanisms in lymphatic endothelium, and provide original potential therapeutic targets. In summary, our findings could translate into innovative approaches to treat lymphatic dysfunctions including lymphedema.